Prostate cancer (PCa) is one of the most heritable human cancers, with inherited risk estimates of up to 60%; however, the underlying inherited genetic architecture is still largely unexplained. Our unique access to a collection of 446 blood specimens from 211 PCa families has enabled an innovative family-based approach to identifying novel disease-susceptibility loci. To date, we have collected targeted sequencing data from a panel of 48 ?cancer risk genes? using samples from all of the PCa families, and whole-exome sequencing (WES) data using samples from a subset of the PCa families. We identified 45 familial loss-of-function and missense mutations in 32 DNA damage response and repair genes. Most of these gene products are known to participate in homology-directed DNA repair (HDR), an error-free type of DNA double-strand break (DSB) repair. DSBs can result from androgen receptor (AR)-induced transcriptional stress and increase cancer risk in androgen- responsive tissues, such as the prostate. In response to androgen stimulation, both TOP2B topoisomerase and LINE-1 endonuclease are recruited to active transcription sites and induce DSBs. Accumulated evidence suggests that loss of HDR will promote other error-prone repair pathways, such as alternative non-homologous end joining, causing mutation accumulation and genomic instability. The mutations we have identified are all germline mutations, representing potential causal genetic factors. We therefore hypothesize that functional deficiency in the HDR pathway, due to loss-of-function and missense mutations in HDR genes, contributes to genomic instability and PCa. We have designed the following three specific aims to test our central hypothesis: 1) To determine the functional defects in DNA damage response and HDR caused by PCa-associated HDR gene mutations; 2) To define the roles of nucleases EXO5 and EXD2 and helicases HFM1 and FANCM in resecting DNA ends at AR-induced, TOP2B-linked DSBs for HDR in prostate cells; 3) To assess the biological significance of AR signaling and the HDR pathway in PCa tumorigenesis. Our strong research team includes Dr. Binghui Shen (contact PI, City of Hope [COH]) and Dr. Xiaochun Yu (mPI; COH), both experts in the fields of DNA damage repair and cancer genetics, and recognized PCa biologists Dr. Zijie Sun (mPI; COH) and Dr. Ming- Fong Lin (co-I; University of Nebraska Medical Center). We will test the role of HDR gene mutations as risk factors for PCa and define the underlying molecular mechanism(s) linking HDR gene mutations to illegitimate DNA repair and PCa development. Completion of the proposed work will significantly advance our understanding of the role of HDR in cancer, especially PCa. We will use an innovative approach to identify novel PCa-associated HDR defects based on familial inheritance and evaluation of both loss-of-function and missense mutations. These insights will impact the field by increasing the availability of biomarkers for early diagnosis and providing molecular targets for anti-cancer drug development.